Nef is an important virulence factor for HIV-1. This protein down-regulates CD4 and class I MHC from the cell surface, and it enhances viral infectivity and replication in vitro. The overall goal of this proposal is to understand the molecular basis of these effects. First, the mechanisms by which Nef modulates the trafficking of CD4 and class I MHC via vesicles coated with the clathrin-adaptor-protein (AP) complexes will be determined. Second, the modulation by Nef of the trafficking of class 1 MHC through a clathrin-independent endosomal compartment will be examined. Third, the roles of CD4, Env, viral entry, and viral uncoating in the enhancement of viral infectivity by Nef will be determined. Several approaches will be used to accomplish these objectives. 1) Specific adaptor complexes will be inhibited using siRNA and/or dominant-negative complex subunits to determine their roles in Nef functions. 2) A variety of in vitro protein interaction assays including a permeabilized cell system will be used to test the hypothesis that Nef regulates the membrane-association of AP complexes by mimicry of the activated form of the cellular protein ARF 1.3) The relationship of Nef to the ARF6-regulated and clathrin-independent endosomal system will be examined using ARF6-mutants and manipulations that alter the organization of this system. A high-efficicncy transfection method will be used to determine whether Nef utilizes this pathway during the down-regulation of class I MHC in T cells. 4) Potential cofactors and processes through which Nef enhances viral infectivity will be examined. These experiments will include: 1) an analysis of viral morphogenesis in T cells in which CD4, gp41, and Gag products will be co-localized in the presence and absence of Nef and in the presence and absence of a mutation in the cytoplasmic domain of gp41 that impairs the incorporation of Env into virions; 2) an evaluation of the role of viral entry using two relatively new assays applied to both the CD4-dependent and CD4-independent effects of Nef; and 3) the development of a viral uncoating assay and its use in testing the hypothesis that Nef facilitates core disassembly in newly infected cells.